To develop a model system in which we could induce GCRs, we used the rare restriction enzyme I-SceI, whose 18 bp recognition sequence is not normally present in the human or mouse genome, to produce a single DNA DSB within a mammalian cell, based on the hypothesis that improper repair of these breaks could lead to GCRs. This enzyme has been used in a series of elegant studies to produce specific, non-random GCRs mediated by homologous recombination in mammalian cells. We generated a construct that expressed the Herpes simplex virus type I thymidine kinase (TK) gene under the control of the constitutive EF1a promoter, with the recognition sequence for the I-SceI restriction enzyme placed between the EF1a promoter and the TK gene. This pEF1aTK vector was introduced into the U937 cell line, and verified that expression of the TK gene conferred sensitivity to ganciclovir (GCV). We then carried out a series of experiments that utilized the negative selection provided by the expression of TK. Cells were transfected with an I-SceI expression vector and selected with GCV (to select for cells that had lost TK expression). Using this system, we reported the discovery of templated sequence insertions, or TSIs in FY2014. Surprisingly, identical TSIs were seen in normal individuals, indicating that most TSIs were polymorphic in the human germline; we termed these TSIPs for TSI polymorphisms. To gain a broader insight into the nature of these TSIPs, we studied a publicly available database of whole genome sequence from 52 individuals, and identified approximately 200 TSIPs. Specific TSIPs tracked with known patterns of human migration. Interestingly, we identified over 20 TSIPs that were derived from mitochondrial DNA, demonstrating that mitochondrial sequences can insert into nuclear sequences, and a subset of TSIPs could be linked to human disease, due to insertions into critical coding regions. These results were published in 2014, 2015, and a third article is currently in press. We were unable to generate GCRs by inducing a single DNA DSB. However, insertion of the EF1aTK vector into H2AX knockout (KO) cells, followed by transfection of an I-SceI expression vector, generated GCR (balanced translocation or megabase inversion) in approximately 15% of GCV resistant clones.. A manuscript describing these findings was published in 2017. An additional manuscript in collaboration with Dr. Nussenzweig is currently in press.